Structural and dynamic properties of juxta-membrane segments of caveolin-1 and caveolin-2 at the membrane interface.

Caveolins (cav1-3) are essential membrane proteins found in caveolae. The caveolin scaffolding domain of cav-1 includes a short sequence containing a CRAC motif (V94TKYWFYR101) at its C-terminal end. To investigate the role of this motif in the caveolin-membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav-1(V94-L102) nonapeptide encompassing this motif and including the first residue of cav-1 hydrophobic domain (L102), in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. Cav-1(V94-L102) partitioned better in DPC and in DM/anionic lipid micelles than in DM micelles, as shown by fluorescence titration and CD. NMR data revealed that this peptide folded as an amphipathic helix located in the polar head group region of DPC micelles. The two tyrosine side-chains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face. Fluorescence studies showed significant Trp subnanosecond rotations, the presence of several rotamers, and a heterogeneous location within the water/micelle interface. NMR studies of the shorter cav-1(V94-R101) peptide and of the homologous sequence of cav-2(I79SKYVMYKF87) allowed the description of the effect of L102 and of the amino acid variations occurring in cav-2 on the structure and localization in DPC micelles. Based on the topological model of caveolins, our results suggest that the cav-1 and cav-2 nonapeptides studied form interfacial alpha-helix membrane anchors in which the K/RhhhYK/Rh motif, also found in cav-3, may play a significant role.

Role of the membrane interface on the conformation of the caveolin scaffolding domain: a CD and NMR study.

Circular dichroism (CD) and NMR spectroscopy were used to study the conformational properties of two synthetic peptides, D82-R101 and D82-I109, encompassing the caveolin scaffolding domain (D82-R101), in the presence of dodecylphosphocholine (DPC) micelles. Our data show that a stable helical conformation of the caveolin scaffolding domain in a membrane mimicking system is only obtained for the peptide including the L102-I109 hydrophobic stretch, a part of the caveolin intra-membrane domain. Through chemical shift variations, an ensemble of six residues of the D82-L109 peptide, mainly located in the V(94)TKYWFYR(101) motif were found to detect the presence of phosphatidylserine solubilized in DPC micelles. Our results constitute a first step for elucidating at a residue level the conformational properties of the central region of the caveolin-1 protein.